Increased mitochondrial superoxide in the brain, but not periphery, sensitizes mice to angiotensin II-mediated hypertension

Figures

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  Fig. 1. MnSOD is only knocked-down in peripheral tissues of the ROSA-Cre tamoxifen inducible mouse model. Mice were treated with tamoxifen (150 mg/kg) daily for 6 days followed by a 14-d incubation period to allow endogenous MnSOD to turnover prior to tissue harvest. A. Representative western blots and quantification of MnSOD protein in various peripheral tissues. B. Representative western blots and quantification of MnSOD protein in regions of the brain. C. Schematic of experimental setup and timeline. *p<0.05 vs. MnSODL/L ROSA-Cre−/−.

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  Fig. 2. Peripheral MnSOD knock-down does not affect baseline or AngII-mediated changes in MAP. Mice were treated with tamoxifen (150 mg/kg) daily for 6 days followed by a 14-d incubation period to allow endogenous MnSOD to turnover prior to subcutaneous AngII infusion (400 ng/kg/min). Daily averages of telemetric MAP data acquired over the course of the experiment. Tam indicates final day of tamoxifen administration. 2-way ANOVA followed by Bonferroni correction post hoc test was utilized for statistical analysis.

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  Fig. 3. MnSOD knock-down in the SFO sensitizes mice to chronic AngII-mediated hypertension. Mice were treated with a one-time ICV injection of either AdCre or AdControl virus followed by a 14-d incubation period to allow endogenous MnSOD to turnover prior to tissue harvest. A. Schematic of experimental setup and timeline. B. Quantitative real-time RT-PCR analysis of MnSOD mRNA from SFO tissue obtained via laser microdissection. *p<0.05 vs. AdControl. C. Daily averages of telemetric MAP data acquired over the course of the experiment. 2-way ANOVA followed by Bonferroni correction post hoc test was utilized for statistical analysis.

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  Fig. 4. MnSOD knock-down in the SFO mice elevates mitochondrial O2•−in response to systemic AngII infusion. Mice were treated with a one-time ICV injection of either AdCre or AdControl virus followed by a 14-d incubation period to allow endogenous MnSOD to turnover prior to subcutaneous AngII infusion (400 ng/kg/min). Tissue was isolated 2 days following AngII infusion. A. Left, representative confocal microscopy images showing DHE fluorescence in the brain SFO from an AdControl or AdCre-injected mouse with or without AngII infusion. Right, quantification of DHE oxidation. B. Left, representative confocal microscopy images showing MitoSOX fluorescence in the brain SFO from an AdControl or AdCre-injected mouse with or without AngII infusion. Right, quantification of MitoSOX oxidation. 2-way ANOVA followed by Bonferroni correction post hoc test was utilized for statistical analysis. Scale bar=100 μm.

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  Fig. 5. Over-expression of MnSOD in the SFO decreases peripheral blood pressure in response to chronic AngII-mediated infusion. Mice were treated with a one-time ICV injection of either AdMnSOD or AdControl virus followed by a 4-d incubation period to allow exogenous MnSOD to be expressed prior to tissue harvest. A. Schematic of experimental setup and timeline. B. Quantitative real-time RT-PCR analysis of MnSOD mRNA from SFO tissue obtained via laser microdissection. *p<0.05 vs. AdControl. C. Representative confocal microscopy images of GFP and MnSOD protein expression in the brain SFO from an AdControl (GFP) or AdMnSOD-injected mouse. D. Daily averages of telemetric MAP data acquired over the course of the experiment. 2-way ANOVA followed by Bonferroni correction post hoc test was utilized for statistical analysis. Scale bar=100 μm.

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  Fig. 6. MnSOD over-expression in the SFO mice decreases mitochondrial O2•−in response to systemic AngII infusion. Mice were treated with a one-time ICV injection of either AdMnSOD or AdControl virus followed by a 4-d incubation period to allow exogenous MnSOD to be expressed prior subcutaneous AngII infusion (400 ng/kg/min). Tissue was isolated 14 days following AngII infusion. A. Left, representative confocal microscopy images showing DHE fluorescence in the brain SFO from an AdControl or AdMnSOD-injected mouse with or without AngII infusion. Right, quantification of DHE oxidation. B. Left, representative confocal microscopy images showing MitoSOX fluorescence in the brain SFO from an AdControl or AdMnSOD-injected mouse with or without AngII infusion. Right, quantification of MitoSOX oxidation. 2-way ANOVA followed by Bonferroni correction post hoc test was utilized for statistical analysis. Scale bar=100 μm.